The goal of this research is to understand the nature of apparently noncovalent changes in globular proteins that occur on ageing. We will study enolase from the nematode T. aceti for which there is substantial evidence that ageing in vivo and in vitro gives rise to a structurally different product. We propose to examine the UV absorption and fluorescence, the circular dichroism, SDS-PAGE behavior, and the equilibrium and kinetic aspects of denaturation and renaturation of this protein. The possibility that oxidative processes are responsible for the observed changes will be examined. The possibility that "old" and "young" enzymes differ in content of metal ions and/or some other low molecular weight species will be tested. We also propose to compare the composition of "old" and "young" proteins, as determined by enzymic hydrolysis followed by chromatographic analysis of the products. Depending on the tractability of the problem, we may also be able to undertake studies of the ageing of other enzymes of T. aceti, and possibly of human erythrocytes and/or rat muscle. The information obtained may make it possible to test the hypothesis that some native proteins are structurally and energentically metastable, and can undergo spontaneous denaturation without covalent alteration. Such a result would have physiological relevance to protein turnover, and practical relevance to enzyme replacement therapy.